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Targeted mass spectrometry analyses of somatic mutations in colorectal cancer specimens using differential ion mobility

ABSTRACT: Identification of K-Ras and B-Raf mutations in colorectal cancer (CRC) is essential to predict patients' response to anti-EGFR therapy and formulate appropriate therapeutic strategies to improve prognosis and survival. Here, we combined parallel reaction monitoring (PRM) with high-field asymmetric waveform ion mobility (FAIMS) to enhance mass spectrometry sensitivity and improve the identification of low-abundance K-Ras and B-Raf mutations in biological samples without immunoaffinity enrichment. In targeted LC-MS/MS analyses, FAIMS reduced the occurrence of interfering ions and enhanced precursor ion purity, resulting in a three-fold improvement in the detection limit for K-Ras and B-Raf mutated peptides. In addition, ion mobility separation of isomeric peptides using FAIMS facilitated the unambiguous identification of K-Ras G12D and G13D peptides. The application of targeted LC-MS/MS analyses using FAIMS is demonstrated for the detection and quantitation of B-Raf V600E, K-Ras G12D, G13D, and G12V in CRC cell lines and primary specimens.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colon

SUBMITTER: Eric Bonneil

LAB HEAD: Pierre Thibault

PROVIDER: PXD047029 | Pride | 2023-11-20

REPOSITORIES: Pride

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			Action	DRS
	1005_FullMS_PRMt6PepBRAFKRAS_CalCur_amol_0_R1.raw	Raw
	1005_FullMS_PRMt6PepBRAFKRAS_CalCur_amol_0_R2.raw	Raw
	1005_FullMS_PRMt6PepBRAFKRAS_CalCur_amol_0_R3.raw	Raw
	1005_FullMS_PRMt6PepBRAFKRAS_CalCur_amol_10000_R1.raw	Raw
	1005_FullMS_PRMt6PepBRAFKRAS_CalCur_amol_10000_R2.raw	Raw

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Project description:The small GTPase KRAS transmits signals from cell surface receptors to various downstream signaling pathways, thereby regulating a multitude of cellular processes. Activating KRAS mutations occur in approximately 30% of human cancers and contribute to malignant transformation through constitutive activation of downstream signaling cascades. These mutations cluster in several hotspots, with codons 12 and 13 being most commonly. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. To address this hypothesis, we used MCF10A human mammary epithelial cells to establish isogenic cell lines that express eight different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological levels, and investigated the biochemical and functional consequences of the different KRAS variants in vitro. In addition, selected effects were evaluated in comparison to cells that overexpress mutant KRAS. The overall effects of mutants expressed at normal levels were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes or migratory abilities. KRAS-G12D, -G12V, -G13D, and -K117N were able to mediate EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by the K117N and Q61H variants. Analysis of known RAS effectors showed that none of the mutations led to increased phosphorylation of ERK, PDK1, or AKT. Both codon 13 mutations were associated with increased EGFR expression, which was not seen with other variants. Finally, comparative gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes, such as enhanced cytokine and p53 signaling, upon G13D expression. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells.

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Targeted mass spectrometry analyses of somatic mutations in colorectal cancer specimens using differential ion mobility

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